The goal of this K08 application is to provide Dr. Julia Carnevale, MD, with the skills she will need to become an independently-funded laboratory investigator studying novel ways to engineer T cells with more potent anti-tumor activity. There is intense clinical interest in improving adoptive T cell therapies to treat and cure more cancers. However, until recently there has been no way to efficiently and comprehensively evaluate the genome to find the critical gene networks that can be leveraged to program favorable human T cell behaviors. Dr. Carnevale recently developed a novel screening technology, sgRNA lentiviral infection with CAS9 electroporation (SLICE), that enables genome-wide CRISPR screening in primary human T cells to search for genes that regulate key therapeutic functions. Dr. Carnevale used SLICE to perform a genome-wide screen in T cells which identified a host of regulators of proliferation, many of which also enhanced T cell activation and in vitro cancer cell killing. She now proposes targeting top-raking genes from this screen, SOCS1 and RASA2 (negative regulators of JAK/STAT and MAP kinase signaling, respectively), to improve the efficacy of adoptive T cell therapies. She hypothesizes that disruption of SOCS1 or RASA2 will enhance the anti-tumor properties of human T cells, that these genes can be targeted to improve adoptive T cell therapies, and that SLICE pooled screening can be performed in vivo to identify modifier gene targets that synergize with SOCS1 and RASA2 loss to improve tumor control. The specific aims of the proposed research are: 1) to test how SOCS1 and RASA2 influence T-cell responses to repeated stimulation and explore the molecular pathways that govern these responses, 2) to determine the effects of SOCS1 or RASA2 inactivation on antitumor activities of T cells in vivo, and 3) to identify gene targets that synergize with SOCS1 or RASA2 loss in vivo. Dr. Carnevale's training and research plans includes a combination of structured coursework and workshops, one-on-one tutorials, and hands-on research experience that will all take place at UCSF, a world-renowned academic center of excellence in basic and translational research. Dr. Carnevale's training plan will complement her existing expertise to build a strong foundation in the following areas: 1) fundamental T cell biology including the study of regulatory circuitry, function, and tumor immunity, 2) preclinical modeling of adoptive T cell therapies, and 3) next-generation sequencing methods and analysis including single-cell RNA sequencing. This project will be conducted under the mentorship of her primary mentor, Dr. Alan Ashworth, President of the UCSF Cancer Center and world expert in therapeutic discovery and translational research, and her co-mentor, Dr. Alex Marson, leading expert in genome engineering in immune cells. She will also receive extensive input from her distinguished advisory panel with complementary expertise to guide her research and career trajectory. At the completion of this award, Dr. Carnevale will have the relevant didactic and research experience to become a leader in discovering and developing next-generation adoptive T cell therapies for cancer patients.